We proposed to continue our work on cellular aging in Dictyostelium discoideum. We are using microtechniques to follow biochemical events during presumptive cell migrations of two major cell types. One cell type undergoes aging and cell death while the other type, although stemming from presumptive cells identical to the aging cells, escapes the aging process and initiates the life cycle again. Our evidence suggests that the aging of stalk cells is under regulation by cyclic 3'-5' AMP. We plan to utilize our experience with microtechniques to follow the biochemical events capable of regulating cyclic AMP levels during the presumptive cell migrations leading to the alternate pathways of aging in the two cell types. The enzymes and metabolites to be followed with the microassays include cyclic AMP, ATP, 5'AMP, adenyl cyclase, cyclic AMP phosphodiesterase, and alkaline phosphatase (5'AMP nucleotidase). We plan to characterize those enzymes or isozymes which are localized in a specific cell type by linking the microtechnique to large scale enzyme purification methods. If during the fractionation procedure more than one form of the enzyme becomes apparent (as we have observed for 5'AMP nucleotidase), we will identify which form corresponds to the enzyme which was localized. To identify the fraction which is localized we will utilize additional microtechniques including capillary tube polyacrylamide gel electrophoresis, electrofocusing in thin-layer polyacrylamide gels, co-elution of micro-columns, and antibody precipitation of enzyme from microsections. By using this approach we can match an enzyme or isozyme activity obtained by the large scale fractionation to its location within a group of aging cells. We will then characterize that fraction for clues as to its mode of regulation in vivo. Where substrate affinity is physiologically significant or effectors of the enzyme are found, we will use the microtechnique to assay the levels of the regulatory molecule in the actual cells where the enzyme is localized. Thus, by studying the biochemical events occurring in a specific group of aging cells, we hope to provide information on the mechanism underlying the alternate pathways of aging in this organism.